Systems and methods for producing bioproducts

ABSTRACT

The present disclosure is directed to systems and methods for sampling and/or controlling the productivity of a bioreactor. The system and methods can include a vessel capable of providing an environment suitable for containing whole broth that can produce the bioproduct, wherein the whole broth contains media and at least one undissolved species, an automated sampling system, a first analytical instrument, and a control system.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/557,913, filed Aug. 30, 2019, which is a continuation of U.S. patentapplication Ser. No. 14/353,753, filed Apr. 23, 2014, now U.S. Pat. No.10,421,939, which is the U.S. National Stage of InternationalApplication No. PCT/US2012/061706, filed Oct. 24, 2012, which waspublished in English under PCT Article 21(2), which in turn claims thebenefit of U.S. Provisional Application No. 61/550,823, filed Oct. 24,2011, U.S. Provisional Application No. 61/584,192, filed Jan. 6, 2012,and U.S. Provisional Application No. 61/603,809, filed Feb. 27, 2012.The prior applications are incorporated herein by reference in theirentirety.

FIELD

The present disclosure is directed to systems and methods for samplingand/or controlling the productivity of a bioreactor.

BACKGROUND

Bioreactors and some chemical reactors are often isolated from theenvironment for many reasons, including for maintaining sterility, toprevent contamination, or for operator or environmental safety.Obtaining samples from such reactors can be difficult because of theseneeds for isolation. However, often the conditions in the reactor cannotbe properly controlled without measuring properties within the reactor.Although some methods exist for determining or estimating the conditionsin the reactor, such conventional methods are generally not able toutilize the information obtained to alter and/or control thebiologically and/or chemically active environments to improve theconditions and product yield of the systems. Accordingly, systems andmethods that provide sampling and control of reactors or other systemswould be desirable.

SUMMARY

In one embodiment, a system for making a bioproduct includes a vesselcapable of providing an aseptic environment suitable for containingwhole broth that can produce the bioproduct. The whole broth containsmedia and at least one undissolved species. The system also contains anaseptic sampling system operably connected to the vessel and capable ofextracting a sample from the vessel. The system also includes at leastone analytical instrument operably connected to the sampling system, theanalytical instrument being configured to measure at least one propertyof the whole broth in the vessel and generating at least one signal inresponse thereto. A control system is provided that in response to theat least one signal generates at least one output signal capable ofcontrolling at least one device that is configured to alter at least oneproperty of the whole broth within the vessel.

In one embodiment, the system further comprises a second analyticalinstrument operably connected to the vessel, the second analyticalinstrument being configured to measure at least one property of thewhole broth in the vessel and generating at least one second signalwhich is sent to the control system.

In some embodiments, the bioproduct is selected from foods, beverages,biofuels, bioenergy, bio-based ethanol, biodiesel, bio-based adhesives,biochemicals, biotherapeutics, biodegradable plastics, and mixturesthereof. In other embodiments, the bioproduct is a biotherapeutic. Instill other embodiments, the bioproduct is a biotherapeutic selectedfrom pharmaceuticals, therapeutic proteins, protein fragments,antibodies, vaccines, and mixtures thereof.

In some embodiments, the vessel is selected from anaerobic fermenters,aerobic fermenters, stirred-tank reactors, adherent bioreactors,wave-type bioreactors, and disposable bioreactors.

In some embodiments, the undissolved species is selected from livecells, dead cells, cell fragments, solid substrates having cells adheredthereto, particles, and mixtures thereof. In other embodiments, theundissolved species is selected from bacteria, yeast, mammalian cells,and e-coli cells.

In some embodiments, the at least one property of the whole broth isselected from media-level properties and cell-level properties. In otherembodiments, the at least one property of the whole broth is selectedfrom pH, dissolved oxygen, osmolality, nutrient concentrations,ammonia/ammonium, lactate/lactic acid, pCO2, electrolytes (such as K+,Ca++, and/or Na+), amino acids, NAD/NADH, impurities, purity,phenotypes, metabolic states, cell health, cell cycle, cell state, cellnumber, and viable cell volume.

In some embodiments, the at least one analytical instrument is selectedfrom pH probes, dissolved oxygen meters, ion-selective electrodes,osmometry, high-performance liquid chromatography, ultra performanceliquid chromatography, gas chromatography, ion chromatography,conductivity, Raman spectroscopy, near infrared spectroscopy, dielectricspectroscopy, fluorometry, ultraviolet/visible spectroscopy, capacitanceprobes, luminescence, redox probes, flow cytometry, hemacytometry,electro-rotation, electrophoresis, dielectrophoresis, and mixturesthereof.

In some embodiments, the device that is configured to alter at least oneproperty of the whole broth is selected from mixing/agitation systems,temperature-control systems, gas pumps, nutrient pumps, product removalsystems, impurity removal systems, pH adjustment systems, and mixturesthereof.

In some embodiments, the aseptic sampling system is an automatic asepticsampling system comprises (a) a sanitizing fluid inlet valve operablebetween an open position and a closed position; (b) a gas inlet valveoperable between an open position and a closed position; (c) a samplecollection valve operable between an open position and a closedposition; (d) a first outlet valve operable between an open position anda closed position; (e) a variable volume reservoir; and (f) a fluid flowpath interconnecting (a)-(e), wherein when (a), (b), and (d) are in theclosed position, (c) can be in the open position to withdraw a samplefrom the enclosed container into the reservoir along a first portion ofthe fluid flow path, wherein when (a), (b), and (c) are in the closedposition, the sample can be discharged from the reservoir along a secondportion of the fluid flow path through (d), and wherein when (a) is inthe open position and (b) and (c) are in the closed position, asanitizing fluid can be introduced into the fluid flow path through (a)to sanitize at least the first portion of the fluid flow path.

In some embodiments, the aseptic sample system extracts a sample fromthe vessel at least once every 8 hours of operation of a bioreactor orother such system, at least once every 6 hours of operation, at leastonce every 4 hours of operation, at least once every 2 hours ofoperation, at least once every 1 hour of operation, at least once every0.5 hours of operation, at least once every 20 minutes of operation, atleast once every 15 minutes of operation, at least once every 10 minutesof operation, and/or at least once every 5 minutes of operation.

In some embodiments, the control system is configured to alter at leastone property of the whole broth within the vessel resulting in animprovement in at least one of bioproduct yield, bioproduct quality,bioproduct purity, bioproduct production rate, reduced cost, reducedenergy consumption, and reduced waste generation, relative to a systemthat is controlled manually. In some embodiments, the aseptic samplingsystem is operably connected to the control system.

In some embodiments, a system for making a bioproduct includes a vesselcapable of providing an environment suitable for containing whole broththat can produce the bioproduct. The whole broth contains media and atleast one undissolved species. The system also contains at least oneanalytical instrument operably connected to the vessel, the at least oneanalytical instrument being configured to measure at least onecell-level property of the whole broth in the vessel and generating atleast one signal in response thereto. A control system is provided thatin response to the at least one signal generates at least one outputsignal capable of controlling a device that is configured to alter atleast one property of the whole broth within the vessel.

The foregoing and other objects, features, and advantages of theinvention will become more apparent from the following detaileddescription, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrations a schematic of one embodiment of the invention.

FIG. 2 illustrates a schematic control system for controlling theoperation of a bioreactor or other such system by analyzing a sample toprovide media and/or cellular level information about the sample.

FIG. 3 illustrates a schematic view of an aseptic autosampling systemfor obtaining samples from enclosed containers.

FIGS. 4A-4D illustrate schematic views of a system for obtaining samplesfrom enclosed containers.

FIGS. 5A-5F illustrate a variable volume reservoir for drawing anddelivery samples from enclosed containers.

FIGS. 6A-6D illustrate schematic views of another system for obtainingsamples from enclosed containers.

FIGS. 7A-7C illustrate enlarged views of exemplary valves that can beused with a sampling system.

FIG. 8 illustrates a cross-sectional view of a system for obtainingsamples from enclosed containers.

FIG. 9 illustrates a partial cross-sectional view of a system forobtaining samples from enclosed containers.

FIG. 10 is an enlarged view of a portion of the system shown in FIG. 5.

FIG. 11 illustrates another embodiment of a system for obtaining samplesfrom enclosed containers.

FIG. 12 illustrates a partial view of a portion of a system forobtaining samples from enclosed containers.

FIG. 13 illustrates a control valve for use with a system for obtainingsamples from enclosed containers.

FIG. 14 illustrates another view of the control valve of FIG. 10.

FIGS. 15A-15D illustrate various views of another embodiment of a systemfor obtaining samples from enclosed containers.

DETAILED DESCRIPTION

Various embodiments of systems and their methods of use are disclosedherein. The following description is exemplary in nature and is notintended to limit the scope, applicability, or configuration of theinvention in any way. Various changes to the described embodiment may bemade in the function and arrangement of the elements described hereinwithout departing from the scope of the invention.

As used in this application and in the claims, the singular forms “a,”“an,” and “the” include the plural forms unless the context clearlydictates otherwise. Additionally, the term “includes” means “comprises.”Further, the term “coupled” generally means electrically,electromagnetically, and/or physically (e.g., mechanically orchemically) coupled or linked and does not exclude the presence ofintermediate elements between the coupled or associated items absentspecific contrary language.

The terms “upstream” and “downstream” are not absolute terms; instead,those terms refer to the direction of flow of fluids within a channel orpathway. Thus, with regard to a structure through which a fluid flows, afirst area is “upstream” of a second area if the fluid flows from thefirst area to the second area. Likewise, the second area can beconsidered “downstream” of the first area.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, percentages,measurements, distances, ratios, and so forth, as used in thespecification or claims are to be understood as being modified by theterm “about.” Accordingly, unless otherwise indicated, implicitly orexplicitly, the numerical parameters set forth are approximations thatmay depend on the desired properties sought and/or limits of detectionunder standard test conditions/methods. When directly and explicitlydistinguishing embodiments from discussed prior art, the embodimentnumbers are not approximates unless the word “about” is recited.

Although the operations of exemplary embodiments of the disclosed methodmay be described in a particular, sequential order for convenientpresentation, it should be understood that disclosed embodiments canencompass an order of operations other than the particular, sequentialorder disclosed. For example, operations described sequentially may insome cases be rearranged or performed concurrently. Further,descriptions and disclosures provided in association with one particularembodiment are not limited to that embodiment, and may be applied to anyembodiment disclosed.

As described herein, various systems and methods are provided forobtaining samples from or measuring properties of a bioreactor (or othersystems that support biologically and/or chemically active environments)and altering the inputs to and/or environment of the bioreactor toadjust the growth or productivity of the media in the bioreactor basedon information obtained. In one embodiment, the information obtainedincludes media level information. In another embodiment, the informationobtained includes cellular level information. In yet another embodiment,the information obtained includes both media level information andcellular level information.

As used herein, bioproducts (also known as bio-based products) arematerials, chemicals, and energy derived from renewable biologicalresources. Examples of bioproducts include foods, beverages, biofuels,bioenergy, bio-based ethanol, biodiesel, bio-based adhesives,biochemicals, biotherapeutics, biodegradable plastics, and mixturesthereof. Specific examples of bioproducts include antibiotics, aminoacids, enzymes, monomers, proteins, food cultures, biopolymers, ethanol,isopropanol, isobutanol, flavorings, perfume chemicals, and the like.

Bioreactors

Bioproducts are made in bioreactors, which are systems that support abiologically active environment. Examples of bioreactors includefermenters (anaerobic or aerobic), stirred-tank reactors, adherentbioreactors, wave-type bioreactors, and disposable bioreactors. Abioreactor can include, for example, a large fermentation chamber forgrowing organisms that can be used to produce bioproducts.

Bioreactors generally contain whole broth. As used herein, the term“whole broth” means the contents of the bioreactor (or a portionthereof), including “media” and “undissolved” species. As used herein,the term “media” means the liquid phase, including all dissolvedsubstances, such as nutrients, dissolved organics, ionic species, etc.As used herein, the term “undissolved” species means the live cells,dead cells, cell fragments, solid substrates having cells adheredthereto, or other particles present in the whole broth. In oneembodiment, the live cells are selected from bacteria, yeast, mammaliancells, and e-coli cells.

In one example, bio-therapeutic proteins can be produced fromgenetically modified mammalian cells within a bioreactor. Suchproduction can be from cell lines of established cell cultures, such as,for example, CHO, NS0, or PER.C6. These cells express the protein ofinterest and subsequently secrete the protein into the media. In manyinstances, mammalian cells are grown in a fed-batch process; however, itshould be understood that the methods and systems disclosed herein canbe applicable in perfusion type cell culture systems.

In some instances, bioreactors can be configured to adjust or controlinputs to the bioreactor, including, for example, one or more of thefollowing variables of pH, dissolved oxygen (DO), reactant/nutrientconcentrations, temperature, and agitation. Such bioreactors can includestirred-tank type reactors, as well as adherent bioreactors, wave-typebioreactors, and disposable bioreactors.

Bioreactors are typically equipped with a means for mixing or agitatingthe whole broth in the bioreactor, including using mechanical mixing,circulation pumps, shifting baffle plates, mechanical vibration schemes,ultrasonic agitation, acoustic agitators, gas bubble agitators, vortexgenerators, cavitation pumping, and combinations thereof. Bioreactorsalso typically are equipped with heat exchangers for maintaining orcontrolling the temperature in the bioreactor.

Analysis of the Whole Broth

The whole broth can be analyzed to provide insight into the contents ofthe bioreactor. The analysis of the whole broth can include adetermination of one or more properties of the media within thebioreactor, a determination of one or more properties of undissolvedspecies (e.g., cells) contained in the whole broth, or both adetermination of one or more properties of the media within thebioreactor and a determination of one or more properties of undissolvedspecies contained within the bioreactor.

Media Level Process Analytical Technologies

A bioreactor can be analyzed for media level information. Such medialevel process analytical technologies (PAT) can be used to measurecertain analytes or properties of the media including, for example, pH,dissolved oxygen (DO), osmolality, glucose or other carbon sources,ammonia/ammonium, lactate/lactic acid, pCO2, electrolytes (such as K+,Ca++, and/or Na+), amino acids, and NAD/NADH concentrations. Bioproductpurity, impurities levels, and other parameters may also be measured.Analytical instruments include pH probes, dissolved oxygen meters,ion-selective electrodes, osmometry, high-performance liquidchromatography, ultra performance liquid chromatography, gaschromatography, ion chromatography, conductivity, Raman spectroscopy,near infrared (NIR) spectroscopy, dielectric spectroscopy, fluorometry,ultraviolet/visible spectroscopy, capacitance probes, luminescence, andredox probes.

Cell Level Process Analytical Technologies

A bioreactor can also be analyzed for cellular properties (part of theundissolved species in the whole broth) which may be indicative of, forexample, cell behaviors, phenotypes, metabolic states, health, and/orcell cycle. Such cellular level PATs can include, for example,dielectric spectroscopy (to determine electrical cell properties), flowcytometry (either incorporating staining of organelles or not),Raman/NIR spectroscopy (which can provide information about cell healthin certain cases), automated hemacytometer, electro-rotation,electrophoresis, and/or dielectrophoresis.

Process Controls Based on Sample Analysis

In the embodiments described herein, a data-rich, cell-level PAT, suchas dielectric spectroscopy or the other measurement tools describedherein, can be used to measure or characterize an aspect of cellbehavior within a sample. In addition, in some embodiments, a data-rich,media-level PAT device can also be used in conjunction with thecell-level PAT. For example, a chemistry analyzer can be provided and atleast a portion of the sample can be directed to the chemistry analyzerto obtain additional information about the media of the sample. Theresult is a system where the interaction of process conditions and cellscan be obtained, thereby enabling process control (as described in moredetail below) based at least in part on cell behavior, rather thantreating the cells as a black-box and controlling only whole brothparameters. In some embodiments, the interaction of process conditionsand cells can be observable on-line and in real time, thereby enablingon-line process control.

FIG. 1 illustrates a schematic of one embodiment of the invention. Here,a vessel is charged with whole broth. An aseptic sampling system removesa sample of whole broth from the vessel and directs it to at least oneanalytical instrument. The analytical instrument generates a signal inresponse to the composition of the sample, the signal sending a signalto a control system. A control system can be provided to control,monitor, and/or communicate with any of the devices/instrumentsdescribed herein. For example, the control system can be configured toperform various tasks, including controlling operation of the samplingsystem, receiving information from analytical instruments, and/orcontrolling the operation of the devices or instruments provided hereinto alter the environment of the bioreactor, Thus, for example, thecontrol system can be configured to receive information from one or moreanalytical instruments and, in response to that received information,send one or more control signals to one or more devices instructing suchdevices to alter at least one property of the whole broth within thevessel.

In some embodiments, the control system can be configured to communicatewith the aseptic sampling system and/or to control operation of theaseptic sampling system. For example, the control system can send one ormore signals to the sampling system to instruct the sampling system toobtain a sample of whole broth and/or to determine when a sample ofwhole broth is removed from the vessel.

In addition to controlling, monitoring, and/or analyzing variousapplications relating to the bioreactor, the control system can beconfigured to display information to a user, including, for example,information about drawing of a sample, information about the analysis ofthe sample (e.g., as provided by the analytical instrument(s)), and/orinformation about the operation of various devices/instruments that arebeing monitored and/or controlled by the control system.

FIG. 2 illustrates an exemplary control schematic for manipulatingvariables in a bioreactor process based on the performance of one ormore analytical instruments to measure a media and/or cellular levelattributes of a sample. As shown in FIG. 3, one or more samples can bedrawn from a vessel, such as a bioreactor by a sampling device, such asan automated sampling device. The sample (or samples) can be analyzed byone or more process analytical technologies (PATs) to identify variousattributes of the media, the cells in the whole broth, and/or both. ThePATs can be performed by one or more devices that are operativelycoupled to the sampling device to perform at-line measurements of thesample(s).

Alternatively, or in addition to the at-line measurements, the PATs candirectly measure the attributes of the media or cells directly in thebioreactor using in situ devices (that is, not through a samplingdevice). The results of these PATs can be provided to a control system(indicated in FIG. 2 as Control Software) and the control system canprovide adjustments to the inputs to the bioreactor and/or to theenvironment within the bioreactor. For example, as shown in FIG. 2,various systems can be provided for controlling variables such asnutrient concentration (e.g., by increasing or decreasing the flow ofnutrients to the bioreactor), DO, pH, or agitation rates. In addition,as shown in FIG. 2, various models can be applied to the control systemto adjust the inputs and/or environment based on an understanding ofcell state trajectory or product quality forecasts that were developedby prior modeling efforts.

A sample can be drawn from the bioreactor and one or more media andcellular level PATs performed on at least a portion of the sample thatis drawn from the bioreactor. In another embodiment, the cellular andmedia level PATs can be measured using an in situ device connected tothe vessel. In one embodiment, the sample can be drawn by an asepticautosampling system such as the system described herein with respect toFIG. 3. However, it should be understood that other sampling devices canbe used to draw a sample from the system, including, for example, anymanual and/or automatic system that is capable of drawing sample fromthe system.

As shown in FIG. 2, a centralized control platform can be provided toreceive the results of the various PATs and to provide real-time processadjustments to the process to alter the environment within thebioreactor in accordance with information obtained from the PATsperformed on the sample.

In one embodiment, the cellular behavior and how it manifests in theprocess variables from the PAT can be characterized in off-line studies.In that manner, the information obtained from the off-line studies canbe used to provide understandings of how the cell states reflected inthe PATs can be used to identify desired changes in the process toprovide improved production and/or yield. For example, theidentification of key biomarkers or causes of impurity formation can bedeveloped in off-line studies and that information can be used toprovide future real-time adjustments to the process. Such off-linestudies can provide relevant modeling information about the currentsample based on previous samples, thereby allowing corrective actions tobe made to the bioreactor in real time.

Accordingly, the new observability provided by the PATs described hereincan be used to create a control model, whereby manipulation ofindividual (or groups) of process variables will affect the outputproduct in a predictable way. This can be accomplished via multi-variatestatistical methods like Multi-Variate Analysis (MVA), or in someexamples, by uni-variate type correlations. After this information isobtained, the control model can run in real-time in a process controlenvironment in order to accomplish the feed-back control as shown inFIG. 2.

The variables manipulated by the centralized control platform can be anyprocess variable or combination of process variables that is foundduring development to affect cell behavior and therefore the product.For example, variables that can be manipulated by the centralizedcontrol platform in response to PATs can include pH, DO, glucose orcarbon source concentration, osmolality, feed flow rate, feedcomposition, temperature, and/or agitation/shear.

In one embodiment, the control system in response to the signalgenerates at least one output signal capable of controlling at least onedevice that is configured to alter at least one property of the wholebroth within the vessel. Devices suitable for this includemixing/agitation systems, temperature-control systems, gas pumps,nutrient pumps, product removal systems, impurity removal systems, pHadjustment systems, and mixtures thereof.

Sampling Systems and Methods

Obtaining samples from bioreactors that support biologically and/orchemically active environments can provide helpful insight about themedia contained with a within the bioreactor. Samples can be obtainedfrom bioreactors in various ways, including some that require manualefforts to draw the sample from the bioreactor and those that areconfigured to automatically obtain the sample. For example, manualsamples can be obtained by directly inserting a sampling device into thebioreactor or otherwise drawing a sample directly from within thebioreactor.

The following description illustrates an exemplary automated samplingsystem that can be used with the systems and methods of producingbiotherapeutics disclosed herein. As described in more detail below, theautomated sampling system disclosed herein can reduce the risk ofcontamination between the drawing of different samples from thebioreactor.

FIG. 3 illustrates a sampling system 100 for obtaining a sample from abioreactor 102 or other similar containers or systems that supportbiologically and/or chemically active environments. Sampling system 100includes a sample collection valve 104 that can open to allow a sampleto enter a fluid flow path 106. The sample can be delivered along theflow path 106 to an outlet valve 108. Outlet valve 108 can open or closeto allow or restrict, respectively, the flow of samples through outletvalve 108. After the sample exits outlet valve 108, the sample can bedirected into an isolated chamber or container 110 for analysis,processing, and/or delivery to another system for analysis and/orprocessing. For example, the sample can be directed from chamber 110 toan automated analyzer 112, such as a bioprofile analyzer available fromNova Biomedical of Waltham, Massachusetts.

The samples that are dispensed from outlet 108 for analysis orprocessing are desirably representative of the materials in bioreactor102 at the time the sample was taken. To reduce the risk ofcontamination, dilution, or alteration of the composition of the samplestaken from sample collection valve 104 and delivered through flow path106, a sanitizing fluid can be delivered through a portion of flow path106 that comes into contact with the samples.

To introduce the sanitizing fluid into flow path 106, a sanitizing fluidinlet valve 114 is provided upstream of sample collection valve 104.Sanitizing fluid inlet valve 114 is operable between a closed positionthat restricts fluid flow through sanitizing fluid inlet valve 114 andan open position that allows fluid flow through sanitizing fluid inletvalve 114. In one embodiment, the sanitizing fluid comprises steam.

A gas inlet valve 116 can also be provided upstream of sample collectionvalve 104 to deliver a gas through flow path 106. The gas can eliminateand/or reduce the amount of sanitizing fluid remaining within flow path106 after flow path 106 is exposed to the sanitizing fluid. Gas inletvalve 116 is operable between a closed position that restricts the flowof gas through gas inlet valve 116 and an open position that allows theflow of gas through gas inlet valve 116. In one embodiment, the gascomprises compressed air.

To draw a sample from bioreactor 102, a variable volume reservoir 118can be provided downstream of sample collection valve 104. Variablevolume reservoir 118 can be moveable between a first position and asecond position to draw a sample through sample collection valve 104 andinto flow path 106. The sample can be drawn into at least a portion ofvariable volume reservoir 118 along a first portion of flow path 106 anddischarged from variable volume reservoir 118 along a second portion offlow path 106. Variable volume reservoir 118 can comprise a diaphragmpump (as shown in FIG. 3), a syringe pump, or other similar devicecapable of drawing a sample from bioreactor 102.

As shown by dotted lines in FIG. 3, at least a portion of samplingsystem 100 can comprise a unitary structure 125. Thus, for example,unitary structure 125 can comprise sample collection valve 104,sanitizing fluid inlet valve 114, gas inlet valve 116, outlet valve 108,and at least a portion of the fluid flow path. Preferably, the entireflow path between the sanitizing fluid inlet valve 114 and the outletvalve 108 is internal to the unitary structure 125.

FIGS. 4A-4D are schematic representations of the operation of samplingsystem 100. As described in more detail below, sampling system 100 canbe inserted into bioreactor 102 and can operate to sanitize or sterilizea flow path from the point of insertion with bioreactor 102 through theclosed pathway of flow path 106. By being able to sanitize or sterilizethe entire path downstream of the insertion point of sampling system 100into bioreactor 102, the possibility of contaminating bioreactor 102and/or the samples captured from bioreactor 102 is reduced.

FIG. 4A illustrates a sanitizing procedure in which a sanitizing fluid120 (e.g., steam) is directed into flow path 106 through an opensanitizing fluid inlet valve 114. As shown in FIG. 4A, sanitizing fluid120 is directed along flow path 106, including along the portions offlow path 106 that are in contact with samples that are drawn frombioreactor 102 and dispensed from flow path 106. For example, sanitizingfluid 120 is directed along flow path 106 past sample collection valve104, through variable volume reservoir 118, and out outlet valve 108. Assanitizing fluid 120 comes into contact with the internal surfaces thatdefine flow path 106, those surfaces are sanitized or sterilized.

Referring now to FIG. 4B, sanitizing fluid inlet valve 114 is closed andgas inlet valve 116 is opened to allow a gas 122 (e.g., air) to enterflow path 106. As shown in FIG. 4B, gas 122 can also be directed alongflow path 106, including along the portions of flow path 106 thatsanitizing fluid 120 contacts. In this manner, any remaining sanitizingfluid 120 can be purged from flow path 106. If desired, a filter 124(e.g., a sterile air filter) can be provided upstream of gas inlet valve116 to ensure that the gas 122 that enters flow path 106 issubstantially free of impurities and/or contaminants.

FIG. 4C illustrates the operation of variable volume reservoir 118 todraw a sample 126 from bioreactor 102 through open sample collectionvalve 104. As shown in FIG. 4C, variable volume reservoir 118 comprisesa diaphragm pump that moves from a first volume to a second, largervolume as illustrated by arrow 128. The enlargement of the volume ofvariable volume reservoir 118 draws a sample through open samplecollection valve 104 and into flow path 106. Variable volume reservoir118 has an inlet 130 and an outlet 132. After sample 126 is drawn intovariable volume reservoir 118, the diaphragm pump moves from the second,larger volume back to a smaller volume as illustrated by arrow 134 inFIG. 4D. The reduction of the volume of variable volume reservoir 118discharges sample 126 through outlet 132 of variable volume reservoir118. Sample 126 is then discharged through outlet valve 108 to becaptured for analysis and/or further processing.

Referring again to FIG. 3, as sample 126 is discharged through outletvalve 108, it can be delivered to chamber 110. To facilitate delivery ofsample 126 to chamber 110, a control valve 136 can be provideddownstream of outlet valve 108. Control valve 136 can be configured toprovide a back pressure to cause sample 126 to be directed into chamber110 and to provide a desired back pressure along flow path 106 tofacilitate the sanitizing process (e.g., FIG. 4A) and the purgingprocess (e.g., FIG. 4B). Control valve 136 can be configured to open toallow the discharge of waste. The discharged waste can include, forexample, sanitizing fluid and purging gas that has traveled along theflow path 106 to sanitize and purge excess sample materials from flowpath 106.

FIGS. 5A-5F illustrate an exemplary operation of a variable volumereservoir 118. FIG. 5A illustrates variable volume reservoir 118 in afirst configuration with a very small volume (e.g. approximately zerovolume). FIG. 5B illustrates a sample being drawn into variable volumereservoir 118 through inlet 130, thereby moving a diaphragm 140 ofvariable volume reservoir 118 in the direction of arrow 138. Diaphragm140 can continue to move in the direction of arrow 138 and expand thevolume of variable volume reservoir 118 until variable volume reservoir118 reaches a second configuration with a larger volume as shown in FIG.5C. As shown in FIGS. 5D, 5E, and 5F diaphragm 140 can then move fromthe second configuration to the first configuration, causing the samplecontained within variable volume reservoir 118 to be discharged throughoutlet 132.

FIGS. 6A-6D illustrate another embodiment of a sampling system 200.Sampling system 200 is generally similar to sampling system 100 and likeelements are identified by similar reference numbers. The maindifferences between sampling system 100 and 200 are illustrated in thevarious figures and described in the related descriptions of thosesystems as included herein.

Sampling system 200 can include a sample collection valve 204, an outletvalve 208, a sanitizing fluid inlet valve 214, and a gas inlet valve216. One or more of these valves can be configured to have a valve stem221 and a sealing member 223. Although FIG. 6A illustrates each of thesevalves as having a valve stem 221 and a sealing member 223, it should beunderstood that the type of valve can vary. The valve stems can beactuated by springs or air, and preferably by a combination of spring-and air-actuation.

FIG. 6A illustrates a sanitizing or sterilizing process. During theprocess shown in FIG. 6A, sample collection valve 204, outlet valve 208,and gas inlet valve 216 are closed with sealing members 223 moved intoengagement with the respective openings associated with those valvesinto flow path 206. Thus, for example, the sealing member 223 of samplecollection valve 204 is engaged with an opening between flow path 206and bioreactor 202 to restrict the passage of material in bioreactor 202from entering flow path 206. At least a portion of the valve stem 221associated with the sample collection valve 204 extends into flow path206, but does not entirely block flow path 206. In this manner,sanitizing fluid can pass across a portion of the sample collectionvalve 204 (and other valves in a similar manner) to sterilize andsanitize the portions of the valve that is in flow path 206. Thus, asshown in FIG. 6A, sanitizing fluid is directed through flow path 206across the closed gas inlet valve 216, across the closed samplecollection valve 204, through the variable volume reservoir 218, acrossthe closed outlet valve 208, and out an open control valve 236.Contaminants and other materials caught up in the sanitizing fluid canalso exit control valve 236.

Referring to FIG. 6B, the sanitizing fluid inlet valve 214 can be closedand gas inlet valve 216 can be opened to deliver a purging gas (e.g.,air) through flow path 206 to remove and/or reduce the presence ofsanitizing fluid within flow path 206. The gas is directed through flowpath 206 across the closed gas inlet valve 216, across the closed samplecollection valve 204, through the variable volume reservoir 218, acrossthe closed outlet valve 208, and out the open control valve 236.

Once the gas purges the remaining sanitizing fluid from flow path 206,both the sanitizing fluid inlet valve 214 and the gas inlet valve 216can close to allow a sample to be drawn into flow path 206. As shown inFIG. 6C, variable volume reservoir 218 draws a sample through the opensample collection valve 204 and into the volume of variable volumereservoir 218. Variable volume reservoir 218 then directs the drawnsample further downstream along flow path 206 towards outlet valve 208as shown in FIG. 6D. Outlet valve 208 can open to allow the sample to bedischarged from flow path 206.

FIGS. 7A-7C illustrate enlarged views of exemplary valves that can beused with the systems disclosed in FIGS. 6A-6C. For example, FIGS. 7Aand 7B illustrate a three-way bypass flow valve that can move between anopen configuration (FIG. 7A) and a closed configuration (FIG. 7B). InFIG. 7A, valve stem 221 is shown extending into flow path 206 withsealing member 223 closing a port 231 (e.g., a gas inlet port, a samplecollection inlet port, a sample collection outlet port) into flow path206. In the closed configuration, fluid can flow past valve stem 221 asshown by arrow 225. One or more sealing rings 233 (e.g., O-rings) can atleast partially surround valve stem 221 to restrict the flow of fluidout of flow path 206 in the area of valve stem 221. In addition, a weephole 235 can be provided to further remove any moisture of other fluidsthat may move past sealing rings 233.

A spring 237 can be provided to bias valve stem 221 towards the closedconfiguration (FIG. 7A) and to ensure that sealing member 223 seatsitself properly with port 231. An air inlet 239 can be provided adjacentvalve stem 221 to move valve stem 221 from the closed configuration(FIG. 7A) to the open configuration (FIG. 7B). Compressed air or otherfluids can be directed through air inlet 239, causing valve stem 221 tomove downward as shown in FIG. 7B. As valve stem 221 moves downward,sealing member 223 moves out of engagement with port 231, allowing fluidto pass through port 231 and enter flow path 206 as shown by arrow 241.

FIG. 7C illustrates a two-way valve that is moveable between a closedconfiguration (not shown) and an open configuration (FIG. 7C). As shownin FIG. 7C, a valve stem 221 with a sealing member 223 can move into anopen configuration in the same manner as that shown in FIG. 7B. Such avalve can be used, for example, with a port 231 that is configured to beopened and closed to allow fluid to flow into the pathway, such as asanitizing fluid inlet port or a waste outlet port.

FIG. 8 illustrates a cross-sectional view of a portion of anotherexemplary sampling system 200, shown with an angled fluid path 206between the sanitizing fluid inlet valve 214 and the outlet valve 208.Sample collection valve 204 extends from a main body 235 of samplingsystem 200 to facilitate coupling of sample collection valve 204 withbioreactor 202 (not shown in FIG. 8).

FIGS. 9 and 10 illustrate views of portions of another exemplarysampling system 200, also having an angled fluid path 206 between thesanitizing fluid inlet valve 214 and the outlet valve 208. As shown inthe enlarged partial cross-sectional view of FIG. 9, when samplecollection valve 204 is in a closed position (e.g., with a sealingmember 223 extending into an opening between the bioreactor and flowpath 206), sanitizing fluid can flow around the end of valve stem 221.Thus, for example, as shown by arrows 255, sanitizing fluid can passaround a portion of sample collection valve 204, thereby improvingsanitization or sterilization of the area adjacent the opening extendinginto the bioreactor.

Moreover, by forming sample collection valve with a sealing member 223that tapers from valve stem 221, the area of contact between sealingmember 223 and the opening can be reduced. To provide improved sealingcharacteristics, in some embodiments, the tip of the valve stem canextend at an angle of greater than 50 degrees from the body of the valvestem and, more preferably at an angle of greater than 70 degrees and,even more preferably at an angle of about 80 degrees.

FIG. 11 illustrates another embodiment of sampling system 200, with avariable volume reservoir 218 integrally formed with the sampling systemstructure. Variable volume 218 comprises a diaphragm pump connected flowpath 206 to draw samples from the bioreactor (not shown) to whichsampling system 200 is coupled.

As described above with respect to FIG. 3, a control valve 136 can beprovided downstream of outlet valve 108. Control valve 136 can beconfigured to provide a desired back pressure along flow path 106 tofacilitate the sanitizing process (e.g., FIG. 4A), the purging process(e.g., FIG. 4B), and/or the sample collection process (e.g., FIG. 4D).For example, during the sanitizing process, it is desirable to keep thesanitizing fluid at a desired temperature for a desired length of time(e.g., if steam is the sanitizing fluid it can be desirable to maintainthe steam at about 121° C.). By providing back pressure via the controlvalve, the temperature within the flow path during the sanitizingprocess can be more easily maintained.

FIGS. 12-14 illustrate an embodiment of a control valve 136 thatcomprises a diaphragm valve. As shown in FIGS. 13 and 14, control valve136 can comprise a diaphragm 191 positioned between two wall members193, 195 to restrict and/or allow flow through the control valve. Forexample, first wall member 193 can comprise an inlet 196 and an outlet197. Movement of diaphragm 191 towards first wall member 193 restrictspassage of fluid through inlet 196 and outlet 197. To provide formovement of diaphragm 191, a control air inlet 199 can be provided onthe opposing second wall member 195. An increase in air pressure atcontrol air inlet 199 causes diaphragm 191 to move towards first wallmember 193, while a decrease in air pressure at control air inlet 199causes diaphragm 191 to move away from first wall member 193. In thismanner, back pressure can be adjusted adjacent the outlet valve of thesampling system as needed or desired.

Referring again to FIG. 12, a holding coil 189 can be provided tocontain a sample during a sample collection processing. Holding coil 189can provide a volume into which a sample can be drawn. In operation, thesample is pumped or drawn into holding coil 189 and then drawn into thechamber from holding coil 189. This can allow larger samples to be drawnand, if the sample drawn is larger than the sample delivered intochamber 110, ensure that the sample delivered into chamber 110 is from acentral region of the drawn sample. By capturing a central portion ofthe sample, the likelihood of that sample being contaminated within theflow path of the sampling system can be further reduced.

FIGS. 15A-15D illustrate various views of an integral sampling system300. As in other embodiments, sampling system 300 includes a samplecollection valve 304, an outlet valve 308, a sanitizing fluid inletvalve 314, a gas inlet valve 316, and a flow path 306 extending alongthese valves. A variable volume reservoir 318 can comprise a diaphragmpump that is configured to draw fluid from a bioreactor through an opensample collection valve 304. Sampling system 300 can be formed of anintegral structure that can be coupled to a bioreactor to drawn samplestherefrom.

As discussed above, the variable volume reservoirs can include adiaphragm pump or other similar structures. FIG. 16 illustrates asampling apparatus 400 that comprises a variable volume reservoir 418.Sampling apparatus 400 can generally function similar to other samplingapparatuses described herein. However, instead of the diaphragm pumpsillustrated in the other embodiments, variable volume reservoir 418 is asyringe-type pump. Thus, by operating the syringe-type pump to increasea volume in variable volume reservoir 418, the sample is drawn throughopen sample collection valve 404, into flow path 406, and into thereservoir of the syringe-type pump. As the volume in the syringe-typepump is decreased, the sample is discharged from the reservoir of thevariable volume reservoir 418 and out the outlet valve 408.

The automated sampling systems described herein can advantageously allowfor more frequent collection of data, reduce sampling variation andhuman error associated with the capturing of samples, and reduce costsby reducing labor requirements associated with manual sampling.

In one embodiment, the aseptic sample system extracts a sample from thevessel at least once in 8 hours, at least once in 6 hours, at least oncein 4 hours, at least once in 2 hours, at least once in 1 hour, at leastonce in 0.5 hours, at least once every 20 minutes, at least once every15 minutes, at least once every 10 minutes, and/or at least once every 5minutes.

Exemplary Applications of Various Systems and Methods Disclosed Herein

As described herein, bioreactor feed strategies are typically developedover a series of experiments using limited empirical off-line data.Since cell physiology dynamically determines the nutrient requirementsof the culture, obtaining the appropriate data over the appropriate timeintervals with which to assess the behavior of the cell population tooptimize the performance of the bioreactor process. The asepticautomatic bioreactor sampling systems and on-line dielectricspectroscopy measurements described herein, coupled with cell-basedbioreactor models, can provide a less invasive monitoring and feedbacksystem, and can be implemented through the use of customized bioreactorcontrol code. Moreover, the systems and methods described herein canshorten development timelines and deliver higher quality processproducing product with a significantly lower cost of goods.

As described in more detail herein, in some embodiments, novelbioreactor monitoring technologies can be applied to bioreactorprocesses and the resulting data can be interpreted into useful processunderstanding which can be leveraged for better control of the process.

The following example is described with reference to FIG. 2, whichillustrates a schematic view of an embodiment of a novel system forproviding online and at-line bioreactor monitoring and feedback toolsfor rapid process development.

Bioreactor System/Analytical Devices—Observability p In someembodiments, observability can be achieved by enhanced sampling (e.g.,10-15× over conventional manual sampling) coupled with measurements atdeeper levels. For example, as shown in FIG. 2, additional at-lineprocess analytical technologies can be employed to generate data whichare data-rich and cell-level (as opposed to pH, DO, Temperature). Thisenhanced data package can provide the substrate for understanding theprocess from the base process levels that most directly affect theproduct.

Sufficient observability of the process can be achieved in variousmanners to facilitate the methods described herein. This observabilitycan be achieved by process analytical technology (PAT) that provides 1)frequent enough data collection to create a robust model of the process,and 2) data from the levels of the process which are most directlyaffecting the product. Such PAT can include, for example, an automatedaseptic sampling (AAS) valve, such as is described herein, anddielectric spectroscopy. The AAS valve can be used to generate morefrequent data at-line and at production scale for cell cultureprocesses.

The AAS valve can be a self-steam sterilizing auto-sampling valve, suchas is described elsewhere herein, which can feed sample to at-line PATat frequencies such as those described above. Depending on which at-linemeasurements are employed, the AAS valve can allow generation of media,cell, and product level information across scales.

Dielectric spectroscopy can be used to illuminate the cell-levelbehavior of the process. This non-invasive method can provide adata-rich cell-level snapshot of the cells in the bioreactor. Usingdielectric spectroscopy, a probe can detect the capacitance, or abilityto store electrical charge, of the cells in suspension as a function offrequency. The resulting capacitance, or dielectric spectrum will beaffected by cell attributes like morphology, membrane charge,organelles, health, and buildup of key metabolites within the cell, andcan therefore yield information about these attributes in real-time ifanalyzed correctly.

Process Understanding and Modeling

In some embodiments, process understanding and data analysis can includethe application of key tools, models, flowcharts to reduce large datasets into useful guidance. The resulting data package can be analyzed insuch a way as to create useful information and process understandingfrom the data. Tools such as multi-variate analysis (MVA), cell- andreactor-level modes, and creative experimentation can be used to greatlysimplify the analysis of this data. The understanding generated fromthis type of analysis can result in something more like a product-scalemodel linking the process parameters, or inputs, to the observed productproperties, or out puts, via the intermediate cell-level observationsgained from the additional PAT. This model, representing an enhancedunderstanding, can then be leveraged to control the bioreactor processfrom a more product-level scope.

In order to turn the potentially daunting amount of data generated fromthe enhanced PAT employed, the novel systems and methods describedherein can turn that data into useful guidance for process monitoringand development. For example, multi-variate data analysis (MVA)techniques, coupled with appropriate process models and subsequentexperimentation, can greatly aid in the reduction of this data intouseful process guidance. Some example tools which can be utilizedinclude, for example, (1) computational fluid dynamic (CFD) models toanswer key production scale vessel design questions such as vent filterplacement to reduce fouling, sparge design, and mass transferoptimization, (2) impeller design guidance based upon bioreactormodeling (e.g., Matlab software-based and CFD commercial software-based)and appropriate experimental verification, (3) predictive cell-basedmodels to address specific product-quality issues, (4) cell/tankmodeling user interface based on kinetic and CFD modeling, (5) CFDmodeling, (6) latent variable modeling (LVM), (7) transport/dimensionalanalysis, and (8) reaction kinetics.

Process Control

As shown in FIG. 2, process control strategies (e.g., as implemented bycontrol software) can apply process understanding to transform theactual bioreactor process. Such strategies can provide improved activecontrol and/or support for development of process strategy. Enhancedprocess understanding generated from this approach can be used to createadditional “handles” with which to control the process from the celllevel. For example, the feed rate of a component (e.g., nutrient orother feeds) can be modified according to the observed cell state, madepossible by interpretation of cell-level data, as illustrated in FIG. 2.

Thus, using the systems described herein, a sample (e.g., a controlvolume) can be analyzed at both the macro level (e.g., fluid models andPATs that provide information about the impact of the reactorheterogeneity and operation) and micro level (e.g., fluid models andPATs that provide experimental data on rate-limiting steps that impactgrowth and productivity). For example, at the micro level, dielectricspectroscopy can be used to provide information about cell populations,individual cells, and organelles. In some embodiments, the size of thesample can be selected based on the smallest volume that contains all ofthe physics that the system is intended to measure.

The systems described herein result in improvements in at least one ofbioproduct yield, bioproduct quality, bioproduct purity, bioproductproduction rate, reduced cost, reduced energy consumption, and reducedwaste generation, relative to a system that is controlled manually or inthe absence of the system described herein.

EXAMPLE 1

A system similar to that shown in FIG. 1 was used to demonstrate theutility of the invention. A bioreactor (3-L Broadley-James reactorvessel, Irvine, Calif.) was coupled to a Nova Bioprofile® FLEX onlineautosampler (Waltham, Mass.) to measure the concentration of glucose inthe bioreactor. In response to the measured concentration of glucose, acontrol system was operably connected to a pump for adding glucose tothe bioreactor.

The procedure used in this example was as follows. First, the bioreactorwas charged with 2 L of media consisting of glucose and pH 7.4 phosphatebuffered saline (PBS) (Sigma-Aldrich, Milwaukee, Wis.). The initialconcentration of glucose in the bioreactor was about 1.3 g/L.

Glucose utilization in the bioreactor was simulated by diluting thecontents of the bioreactor by adding 550 mL/hr of PBS. Samples from thebioreactor were analyzed every hour using the autosampler. Theautosampler valve works by running a sanitizing solution through thelines, followed by a flush solution (available from Nova Biomedical).The sample is then collected, and sent to the analyzer for glucoseanalysis.

The system is also equipped with a control system that is operablyconnected to a pump for adding glucose to the bioreactor. Using themeasured glucose concentration in the bioreactor, the control systemoperates a glucose pump that is connected to a glucose reservoircontaining a concentration of 300 g/mL in PBS. The control system turnsthe pump off or on depending on the measured glucose concentration. Inthis example, the target glucose concentration was set at 1 g glucose/Lin the bioreactor.

The results of Example 1 are presented in Table 1, which shows thatsampling every hour allowed the system to achieve steady-state (±0.1 g/Lof the set point) after about 4 hours of operation.

TABLE 1 Glucose Concentration versus Time for Example 1 Time (hr)Glucose Concentration (g/L) 0 1.33 1 0.50 2 2.02 3 1.40 4 0.96 5 1.06 60.99 7 0.96 8 1.02 9 1.07 10 0.97 11 0.99 12 1.00 13 1.03

As a control, the system used in Example 1 was operated such that thesampling occurred once every 4 hours. In addition, the initialconcentration of glucose in the bioreactor was 1.24 g/L. The results ofthese tests are shown in Table 2, which shows that it took at least 16hours to achieve steady state operation.

TABLE 2 Glucose Concentration versus Time for Control 1 Time (hr)Glucose Concentration (g/L) 0 1.24 4 0.91 8 0.71 12 1.28 16 0.96 20 1.00

In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated embodiments are only preferred examples of the invention andshould not be taken as limiting the scope of the invention. Rather, thescope of the invention is defined by the following claims. We thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

We claim:
 1. A method of making a bioproduct comprising: extracting a sample from a vessel containing whole broth that can produce the bioproduct, wherein the sample is extracted using an automated sampling system and the whole broth contains media and at least one undissolved species; delivering the sample to an analytical instrument; measuring at least one property of the whole broth in the sample with the analytical instrument; generating at least one first signal in response to the measured at least one property; generating at least one output signal that controls at least one device that is configured to alter at least one property of the whole broth within the vessel, the at least one output signal being generated in response to the at least one first signal; and altering the at least one property of the whole broth within the vessel by the at least one device; wherein the automated sampling system is operably connected to the vessel and capable of extracting the sample from the vessel at predetermined intervals, and wherein the undissolved species being selected from the group consisting of live cells, dead cells, cell fragments, solid substrates having cells adhered thereto, and mixtures thereof.
 2. The method of claim 1, wherein the automated sampling system comprises a variable volume reservoir that includes a reservoir, a reservoir inlet, and a reservoir outlet that is different from the reservoir inlet, and extracting the sample from the vessel comprises: delivering the sample into the reservoir through the reservoir inlet; and discharging the sample from the reservoir by reducing a volume of the reservoir to cause the sample to exit the reservoir through the reservoir outlet.
 3. The method of claim 2, wherein the variable volume reservoir comprises a movable member and a housing, the movable member being configured to move between a first position where the reservoir has a first volume to draw the sample into the reservoir through the reservoir inlet and a second position where the reservoir has a second, smaller volume to discharge the sample from the reservoir through the reservoir outlet.
 4. The method of claim 3, wherein the variable volume reservoir is a flexible diaphragm.
 5. The method of claim 3, wherein the variable volume reservoir is a syringe pump.
 6. The method of claim 1, wherein the bioproduct is selected from the group consisting of foods, beverages, biofuels, bioenergy, bio-based ethanol, biodiesel, bio-based adhesives, biochemicals, biotherapeutics, biodegradable plastics, and mixtures thereof.
 7. The method of claim 1, wherein the bioproduct is a biotherapeutic.
 8. The method of claim 7, wherein the biotherapeutic is selected from the group consisting of pharmaceuticals, therapeutic proteins, protein fragments, antibodies, vaccines, and mixtures thereof.
 9. The method of claim 1, wherein the vessel is selected from the group consisting of anaerobic fermenters, aerobic fermenters, stirred-tank reactors, adherent bioreactors, wave-type bioreactors, and disposable bioreactors.
 10. The method of claim 1, wherein the undissolved species comprises one or more selected from the group consisting of bacteria, yeast, mammalian cells, and e-coli cells.
 11. The method of claim 1, wherein the at least one property of the whole broth comprises media-level properties.
 12. The method of claim 1, wherein the at least one property of the whole broth comprises cell-level properties.
 13. The method of claim 1, wherein the at least one property of the whole broth is selected from the group consisting of pH, dissolved oxygen, osmolality, nutrient concentrations, ammonia/ammonium, lactate/lactic acid, pCO2, electrolytes (such as K+, Ca++, and/or Na+), amino acids, NAD/NADH, impurities, purity, phenotypes, metabolic states, cell health, cell cycle, cell state, cell number, and viable cell volume.
 14. The method of claim 1, wherein the analytical instrument is selected from the group consisting of pH probes, dissolved oxygen meters, ion-selective electrodes, osmometry, high-performance liquid chromatography, ultra performance liquid chromatography, gas chromatography, ion chromatography, conductivity, Raman spectroscopy, near infrared spectroscopy, dielectric spectroscopy, fluorometry, ultraviolet/visible spectroscopy, capacitance probes, luminescence, redox probes, flow cytometry, hemacytometry, electro-rotation, electrophoresis, dielectrophoresis, and mixtures thereof.
 15. The method of claim 1, wherein the device that alters the at least one property of the whole broth is selected from the group consisting of mixing/agitation systems, temperature-control systems, gas pumps, nutrient pumps, product removal systems, impurity removal systems, pH adjustment systems, and combinations thereof.
 16. The method of claim 1, wherein the automated sampling system extracts a sample from the vessel at least once every 4 hours.
 17. The method of claim 1, wherein the automated sampling system extracts a sample from the vessel at least once every 2 hours.
 18. The method of claim 1, wherein the automated sampling system extracts a sample from the vessel at least once every 1 hour.
 19. The method of claim 1, wherein the automated sampling system extracts a sample from the vessel at least once every 0.5 hours.
 20. The method of claim 1, wherein altering the at least one property of the whole broth within the vessel results in an improvement in at least one of bioproduct yield, bioproduct quality, bioproduct purity, and bioproduct production rate. 